Salmonellosis of swine is one of the most economically important of the enteric and septicemic diseases affecting young pigs, and has been described as a significant health problem in swine. Although many serotypes of Salmonella have been isolated from pigs, S. choleraesuis var. kunzendorf and S. typhimurium are the two most frequently isolated serotypes associated with clinical salmonellosis in swine (Wilcock, 1986). S. choleraesuis is host-adapted to swine and is often the etiologic agent of fatal septicemic disease with little involvement of the intestinal tract. This S. choleraesuis reservoir in swine is a concern not only because of its disease-causing potential for pigs, but also because of its public health significance for humans.
The disease caused by S. choleraesuis manifests in many clinical signs. The organism is inherently invasive, and does not require the massive luminal proliferation required by S. typhimurium. Lesions and necrosis occur in the submucosa and lamina propria of the gut. Mortality is high, and the duration and severity of the disease is unpredictable.
Currently there is little information on vaccine use for control of swine salmonellosis. A rough variant of S. choleraesuis was used by H. W. Smith (1965) to demonstrate protection after challenge with virulent S. choleraesuis. However, the animals developed fever, sublethal disease, and became shedders of the bacteria. The Smith strain is commercially available in Europe as Suscovax.TM., which is manufactured and distributed by Wellcome Laboratories. Hanna et al (1979) reported use of Smith's life attenuated S. choleraesuis vaccine by intramuscular route in pregnant sows. The piglets, after birth, had high titers of circulating maternally-derived antibodies, and resisted intranasal challenge. Although it has been reported that galE mutants of S. typhimurium are avirulent and immunogenic, in contrast, S. choleraesuis strains with galE mutations remain as virulent in mice as the wild-type gal.sup.+ parent. S. choleraesuis auxotrophs with requirements for aromatic amino acids due to an aroA mutation have reduced virulence in mice, but were unable, even after three immunizing doses, to induce protective immunity. At present, the only S. choleraesuis vaccine licensed for use in the United States of America is a killed bacterial bactrin, which is not particularly effective in inducing protective immunity.
In general, previously used vaccines against infectious diseases have generally comprised (I) specific components purified from the etiologic agents, including intact antigens, fragments thereof, or synthetic analogs of naturally occurring antigens or epitopes, (II) antiidiotypic antibodies, (III) the whole killed etiologic agent, or (IV) an avirulent derivative of the etiologic agent as a live vaccine. Numerous vaccines of these types exist, of which the following are selected examples:
U.S. Pat. No. 4,250,262 discloses methods for recovering the enzyme glucosyltransferase from Streptococcus mutans and the use of this purified enzyme in local immunization against dental caries, a Type I vaccine. Details for culturing the bacteria, purifying the enzyme, and using the enzyme to stimulate IgA antibody in saliva are presented for serotype a, c or g of S. mutans. Other examples of vaccines from purified specific components of bacteria are found in U.S. Pat. Nos. 4,203,971 and 3,239,749, which disclose a vaccine useful against infection by Neisseria gonorrhoeae which consists of a glycoprotein from the outer coat material of gonococci. Injection of the glycoprotein stimulates a bactericidal antibody.
The use of dead S. mutans cells to immunize against tooth decay via administering in the mouth, which is disclosed in U.S. Pat. No. 3,931,398, is an example of a Type III vaccine. The inventors recognized that immunoglobulin A (IgA) antibodies were the antibodies being produced and that they resulted in a decrease in plaque formation.
A live bacterial vaccine (Type IV) which contains selected strains of Escherichia coli bacteria is disclosed in U.S. Pat. No. 3,975,517. The bacteria were treated with dilute formalin to attenuate or partially inactivate them before injection into the mammary gland of a sow. Antibody thereby produced was later found in the milk and protected newborn swine against E. coli infections. The formalin treatment that caused the E. coli inactivation was only a temporary attenuation of the bacteria and care had to be taken to prevent bacterial recovery before injection. Such recovery would have resulted in serious infection rather than protection.
It has been possible to develop avirulant strains by the introduction of mutations into said strains which results in the strains being substantially incapable of survival in a host. These strains can be said to be genetically attenuated. For ease of description the principles of genetic attenuation will be illustrated by reference to the Salmonella system, however, the principles are broadly applicable as will be discussed below.
Salmonella typhimurium, S. typhi and other Salmonella species with invasive properties enter deep tissues after oral ingestion by attaching to, invading and proliferating in the cells of the gut-associated lymphoid tissue (GALT; Peyer's Patches) (Carter and Collins, J. Exp. Med. 139:1189-1203, (1974)). Since Salmonella-mediated delivery of an antigen to the GALT elicits a generalized secretory immune response as well as humoral and cellular immune responses, avirulent Salmonella mutants that have lost the ability to cause disease without impairment in their ability to attach to and invade the GALT are likely to serve as effective vectors to deliver foreign antigens, such as colonization or virulence antigens, to the GALT and to induce protective immunity against the pathogen from which such antigens were derived. The construction of avirulent Salmonella vaccine strains that express antigens from other microorganisms has been accomplished via classical gene transfer procedures (Formal et al., Infect. Immun. 34:746-750, (1981)) as well as by using recombinant DNA technologies. In the latter case, avirulent Salmonella strains have been constructed expressing genes from organisms that normally exchange genetic information with Salmonella (Stevenson and Manning, FEMS Microbiol. Lett. 28:317-321 (1985); Clements et al., Infect. Imm. 53:685-692, (1986)), as well as from microorganisms that are unable to transfer genetic information to Salmonella by classical means of gene transmission (Curtiss, J. Dent. Res. 65:1039-1045 (1986)). Such bivalent avirulent Salmonella strains have been shown to elicit antibodies and in one instance a cellular immune response (Brown et al., J. Infect. Dis. 155:86-92 (1987)) to the expressed antigen, but data pertaining to induction of protective immunity against the pathogen supplying the colonization or virulence antigen is still scant.
Bacon et al, (Brit. J. Exp. Pathol. 32:85-96, (1951)) were first to investigate the avirulence of auxotrophic mutants of S. typhi. They noted that mutants with requirements for purines, p-aminobenzoic acid, and aspartate had reduced virulence for mice. Germanier and Furer, (Infect. Immun. 4:663-673, (1971)) first investigated use of galE mutants of S. typhimurim for avirulence and immunogenicity in mice and then proposed use of the S. typhi galE mutant Ty21a as a vaccine against typhoid fever in humans (J. Infect. Dis 131:553-558 (1975)). Stocker (U.S. Pat. No. 4,550,081) employed transposon mutagenesis with Tn10 followed by selection for fusaric acid resistance that leads to deletional loss of Tn10 and adjacent DNA sequences to yield deletion mutations unable to revert, a problem that was apparent in the mutants used by Bacon et al. Stocker initially isolated aroA deletion (delta) mutants impaired in the ability to synthesize the aromatic amino acid family of compounds, including p-aminobenzoic acid needed for folate biosynthesis and dihydroxybenzoic acid, a precursor to enterochelin, and more recently combined the delta-aroA mutation with a deletion mutation abolishing adenine biosynthesis. Tn10-induced and fusaric acid resistance-generated delta-asd mutations that impose a requirement for diaminopimelic acid have been employed to render S. typhimurium avirulent without impairing its ability to induce a generalized secretory immune response. Since many Salmonella possess a plasmid that contributes to virulence, plasmid-cured derivatives have been investigated and proposed for use as live vaccines (Nakamura et al, Infect. Immun. 50:586-587 (1985)).
Although each of the above described means for rendering Salmonella avirulent without impairing immunogenicity has merit, each has problems. galE mutants are difficult to grow to maintain immunogenicity, since they are galactose-sensitive but must be grown in the presence of galactose to produce normal LPS which, however, selectively leads to galactose-resistant variants that are non-immunogenic. Although delta-aroA mutants abolish the synthesis of both enterochelin and folic acid, the necessity of enterochelin for S. typhimurium virulence has been called into question by the extensive results of Benjamin et al. (Infect. Immun. 50:392-97 (1985)) that demonstrate that any and all mutations that interfere with S. typhimurium's ability to chelate and transport iron are without significant effect on virulence. Therefore, the avirulence of delta-aroA mutants is most likely solely due to the inability to synthesize p-aminobenzoic acid (pABA) and therefore folic acid. Since Bacon et al. (supra) observed that administering p-aminobenzoic acid in the diet of mice infected with mutants unable to synthesize pABA led to wild-type levels of virulence, one must be concerned with phenotypic reversal of avirulence in vaccine strains due to dietary consumption of metabolites whose synthesis is blocked in the avirulent mutants. The delta-asd mutants, although not having some of these other difficulties, rapidly die following oral feeding and invasion of the GALT, and are thus only effective at eliciting a generalized mucosal immunity and are ineffective in inducing humoral and cellular immunity.
This invention addresses many of the deficiencies of the prior art vaccines by employing transposon-induced mutants in which the impairment leading to avirulence cannot be repaired by diet or by anything the animal host could supply. These deletions could, of course, be introduced by recombinant DNA techniques.